The present invention relates to pharmaceutical compositions containing as active principle an oligosaccharide of formula: 
or to a mixture of these oligosaccharides, to the novel oligosaccharides of formula (I), to mixtures thereof and to methods for their preparation.
In formula (I), n is an integer from 0 to 25, R1, R3, R4, R5, R6 and R8, which may be identical or different, represent a hydrogen atom or an SO3M radical, R2 and R7, which may be identical or different, represent a hydrogen atom or an SO3M or COCH3 radical, and M is sodium, calcium, magnesium or potassium.
These oligosaccharides thus comprise an even number of saccharides.
In formula (I), R4 and R6 are, preferably, hydrogen atoms.
Oligosaccharides of formula (I) for which n is equal to 0, and either R1, R6 and R8 represent a hydrogen atom, R7 represents an SO3M or COCH3 radical and M is sodium, or R1 and R6 represent a hydrogen atom, R7 represents a COCH3 radical, R8 represents an SO3M radical and M is sodium, or R6 represents a hydrogen atom, R1, R7 and R8 represent an SO3M radical and M is sodium have already been described by G. H. LEE et al., J. Chromat. 212, 65-73 (1981), but no pharmacological property is described for these products.
Oligosaccharides of formula (I) for which n is equal to 0, and either R6 and R7 represent hydrogen atoms, R1 and R8 represent an SO3M radical and M is sodium, or R1, R6 and R7 represent a hydrogen atom, R8 represents an SO3M radical and M is sodium, are described by M W McLEAN et al., Eur. J. Biochem., 1984, 145, 607, without any indication of pharmacological activity.
The pharmaceutical compositions which are preferred are those containing an oligosaccharide of formula (I) for which:
n is an integer from 0 to 10, and in particular from 0 to 6, and even more particularly from 1 to 6.
R1, R2, R3, R5, R7 and R8 are identical or different, and represent a hydrogen atom or an SO3M radical, and in particular R1, R2, R3, R5, R7 and R8 are SO3M radicals,
M is sodium.
The pharmaceutical compositions which are particularly preferred are those containing an oligosaccharide of formula (I) for which:
n is equal to 0, R1, R7 and R8 represent an SO3M radical, R6 represents a hydrogen atom and M is sodium,
n is equal to 1, R1, R2, R3, R5, R7 and R8 represent an SO3M radical, R4 and R6 represent a hydrogen atom and M is sodium,
n is equal to 2, R1, R2, R3, R5, R7 and R8 represent an SO3M radical, R4 and R6 represent a hydrogen atom and M is sodium,
n is equal to 3, R1, R2, R3, R5, R7 and R8 represent an SO3M radical, R4 and R6 represent a hydrogen atom and M is sodium,
n is equal to 4, R1, R2, R3, R5, R7 and R8 represent an SO3M radical, R4 and R6 represent a hydrogen atom and M is sodium.
The oligosaccharides of formula (I), with the exception of those for which n is equal to 0 and either R1, R6 and R8 represent a hydrogen atom, R7 represents an SO3M or COCH3 radical and M is sodium, or R1, and R6 represent a hydrogen atom, R7 represents a COCH3 radical, R8 represents an SO3M radical and M is sodium, or R6 represents a hydrogen, R1, R7 and R8 represent an SO3M radical and M is sodium, or R6 and R7 represent hydrogen atoms, R1 and R8 represent an SO3M radical and M is sodium, or R1, R6 and R7 represent a hydrogen atom, R8 represents an SO3M radical and M is sodium, are novel and, as such, form part of the invention.
The oligosaccharides of formula (I) can be prepared by reaction of an alkali metal borohydride or a quaternary ammonium borohydride with oligosaccharides of formula: 
in which n is an integer from 0 to 25, R1, R3, R4, R5, R6 and R8, which may be identical or different, represent a hydrogen atom or an SO3M radical, R2 and R7, which may be identical or different, represent a hydrogen atom or an SO3M or COCH3 radical, and M is sodium, calcium, magnesium or potassium.
This reaction is carried out in aqueous medium, at a temperature in the vicinity of 25xc2x0 C., at a pH between 7 and 10, and preferably between 9 and 10, for the entire duration of the reaction. The pH is maintained by addition of a sodium hydroxide solution at 0.5 mol/l. The reaction is stopped by acidification of the reaction medium, for example by addition of acetic acid until a pH between 4 and 5 is obtained.
As alkali metal borohydrides, mention may be made of lithium borohydride, sodium borohydride and potassium borohydride.
As a quaternary ammonium borohydride, mention may be made of tetrabutylammonium borohydride.
The oligosaccharides of formula (II) can be obtained by gel chromatography separation of a mixture of oligosaccharides (III) obtained by enzymatic depolymerization of heparin or basic depolymerization of the benzyl ester of heparin or of a benzyl ester of semi-synthetic heparin.
This chromatography is carried out on columns filled with gel of polyacrylamide-agarose type, such as that sold under the trade mark Ultrogel ACA202R (Biosepra). Preferably, an array of polyacrylamide agarose gel columns is used. The number of columns used is adapted as a function of the volume, the gel and the oligosaccharides to be separated. The mixture is eluted with a solution containing a phosphate buffer and sodium chloride. Preferably, the phosphate buffer solution is a solution containing 0.02 mol/l of NaH2PO4/Na2HPO4 (pH 7) containing 0.1 mol/l of sodium chloride. Detection of the various fractions is carried out by UV spectrometry (254 nm) and ionic spectrometry (IBF). The fractions thus obtained can then be optionally purified, for example by SAX (strong anion exchange) chromatography according to the methods known to those skilled in the art and in particular according to the methods described by K. G. Rice and R. J. Linhardt, Carbohydrate Research 190, 219-233 (1989), A. Larnkjaer, S. H. Hansen and P. B. Ostergaard, Carbohydrate Research, 266, 37-52 (1995) and in patent WO 90/01501 (Example 2). The fractions are then lyophilized, and then desalified on a gel-filled column such as a column of Sephadex G10(copyright) gel (Pharmacia Biochemicals).
When the purification is not carried out by SAX chromatography, the lyophilizates can be optionally purified by simple or fractional precipitation according to the methods known to persons skilled in the art and in particular according to the method described in patent FR 2 548 672. Generally, the process is performed according to the following procedure:
The lyophilized fraction to be purified is dissolved, at 25xc2x0 C., in about ten volumes of distilled water. On adding methanol or ethanol, the desired oligosaccharide is precipitated, while monitoring its enrichment by HPLC chromatography (high performance liquid chromatography). The addition of methanol or ethanol is determined as a function of the desired yield and purity of said oligosaccharide. Similarly, this operation can be carried out in several successive steps starting with the initial solution of lyophilizate. For this, more of the insolubilizing agent (methanol or ethanol) is added portionwise and the precipitate obtained after each addition is isolated. The precipitates thus prepared are analyzed by HPLC chromatography. Depending on the desired yield and purity, the suitable fractions of precipitate are combined.
According to a variant of the present invention, the lyophilized fraction to be purified can be dissolved in 10 to 200 volumes of water containing from 0 to 30% sodium acetate. The percentage of sodium acetate will be preadjusted as a function of the nature of the oligosaccharide to be treated (a function of the size). On adding methanol, the desired oligosaccharide is precipitated while monitoring its enrichment by HPLC chromatography (high performance liquid chromatography). The addition of methanol is determined as a function of the desired yield and purity of said oligosaccharide. Similarly, this operation can be carried out in several successive steps starting with the initial solution of lyophilizate. For this, more of the insolubilizing agent (methanol) is added portionwise and the precipitate obtained after each addition is isolated. The precipitates thus prepared are analyzed by HPLC chromatography. Depending on the desired yield and purity, the suitable fractions of precipitate are combined.
For the intermediates of formula (II) for which n=0, 1 or 2, it is preferable to start with a mixture (III) obtained by enzymatic depolymerization.
This depolymerization is carried out by means of heparinase I (EC 4.2.2.7), in a pH 7 phosphate buffer solution, in the presence of sodium chloride and of BSA (bovine serum albumin), at a temperature between 10 and 18xc2x0 C., and preferably 15xc2x0 C., for 8 to 10 days, and preferably 9 days. The depolymerization is stopped, for example, by heating the reaction medium at 100xc2x0 C. for 2 minutes, and the mixture is recovered by lyophilization. It is preferable to use 7 IU of heparinase I per 25 g of heparin. The phosphate buffer solution generally comprises 0.05 mol/l of NaH2PO4/Na2HPO4 (pH 7) in the presence of 0.1 mol/l of sodium chloride. The BSA concentration is generally 2%.
For the intermediates of formula (II) for which n=0, 1, 2, 3 or 4, it is preferable to start with a mixture (III) obtained by depolymerizing a benzyl ester of heparin.
The benzyl ester of heparin can be prepared according to the methods described in patents U.S. Pat. No. 5,389,618, EP 40 144 and FR 2 548 672. The degree of esterification will preferably be between 50 and 100%. Preferably, it will be between 70 and 90%.
The depolymerization is carried out in aqueous medium, by means of an alkali metal hydroxide (for example lithium hydroxide, sodium hydroxide, potassium hydroxide or cesium hydroxide) or of a quaternary ammonium hydroxide (for example tetrabutylammonium hydroxide), preferably at a molarity between 0.1 and 0.2 mol/l, at a temperature between 40 and 80xc2x0 C., for 5 to 120 minutes. In one preferred mode, the process is performed for 5 to 15 minutes, at a temperature between 60 and 70xc2x0 C., with a 0.15 mol/l sodium hydroxide solution. The depolymerization reaction is stopped by neutralization by addition of an acid such as acetic acid. After addition of 10% by weight per volume of sodium acetate, the oligosaccharide mixture is precipitated by adding methanol, preferably 2 volumes per 1 volume of reaction medium, and filtered.
According to one preferred aspect of the invention, the oligosaccharide mixture obtained after chemical depolymerization, in the form of an aqueous solution, is enriched by ultrafiltration through membranes with a suitable nominal cut-off threshold (of the Pellicon type made of regenerated cellulose, sold by Millipore); the type of membrane being adapted as a function of the type of enriched oligosaccharides to be recovered. For the oligosaccharides (II) for which n=0, a membrane with a nominal cut-off threshold of 1 kDa will be used, for the oligosaccharides (II) for which n=1, a 1 kDa or 3 kDa membrane will be used, for the oligosaccharides (II) for which n=2, a 3 kDa membrane will be used, for the oligosaccharides (II) for which n=3 or 4, a 5 kDa membrane will be used. During this operation, the permeate is recovered and the retentate is discarded. Thus, the fraction of enriched product can represent from 50 to 10% of the initial oligosaccharide mixture, while at the same time conserving at least 80% of the desired oligosaccharide.
For the intermediates of formula (II) for which n=0 to 25, it is preferable to start with a mixture (III) obtained by depolymerizing a benzyl ester of semi-synthetic polysaccharide sulfate. The benzyl ester of semi-synthetic polysaccharide sulfate is prepared from semi-synthetic polysaccharide sulfates obtained from polysaccharide K5, and according to the methods described in patents WO 94/29352 and WO 96/14425. The esterification, depolymerization and recovery conditions are the same as those described above for the benzyl ester of heparin.
In all the preceding processes, the initial heparin can be of porcine, ovine, caprine or bovine origin and can be obtained from the mucus, lungs or hides of the animals. Preferably, a heparin from porcine or ovine mucus or from bovine lung is used, and even more preferably from porcine mucus or from bovine lung.
The oligosaccharides of formula (I) have anti-inflammatory properties and can thus be used for preventing or treating diseases associated with an inflammatory process involving the production of cytotoxic substances such as nitrogen monoxide (NO), whose inducible form is released in particular by neutrophils or macrophages when these neutrophils or macrophages migrate and are activated in a tissue. The migration, activation and adhesion of neutrophils takes place in tissue regions which have been made ischemic following an occlusion or a spasm of an artery which vascularizes this tissue. These ischemias can arise either in the brain (cerebrovascular accident) or in the myocardium (myocardial infarction) or in the lower limbs (known as peripheral ischemias). The oligosaccharides of formula (I) can thus be used for preventing and/or treating neurodegenerative diseases for which cerebral inflammation plays a deleterious role which can lead to death, among which mention may be made of cerebral ischemias, cardiac ischemias (myocardial infarction), peripheral ischemias, traumas of the central nervous system and in particular cranial, spinal and craniospinal traumas, multiple sclerosis, neuropathic pain and peripheral neuropathies, motoneuron diseases including amyotrophic lateral sclerosis, progressive spinal atrophy, infantile muscular atrophy and primary lateral sclerosis, neuro-AIDS, Alzheimer""s disease, Parkinson""s disease and Huntington""s chorea and certain forms of osteoarthritis, in particular with articular localization.
The anti-inflammatory activity of these products is demonstrated in vivo in the test of production of NOx (nitrite and nitrate) induced by a lipopolysaccharide (LPS) originating from E. coli, according to the protocol described by M. YAMASHITA et al., Eur. J. Pharmacol, 338, 2, 151-158 (1997) or J. E. SHELLITO et al., Am. J. Respir. Cell Mol. Biol., 13, 1, 45-53 (1995).
0.5 mg/kg of the oligosaccharide is injected into male CD1 mice (Charles River, 25-35 g) at T0 via intravenous bolus, and 1 or 2 mg/kg of the oligosaccharide are injected subcutaneously at T+15 minutes. At T+30 minutes, 100 mg/kg of lipopolysaccharide (LPS) originating from E. coli (Sigma L3129, serotype 0127:B8) are administered. At T+3 hours 1 or 2 mg/kg of the oligosaccharide are again injected subcutaneously. At T+5 hours 30 minutes, a blood sample is collected by ocular puncture, and the concentrations of NOx (nitrite and nitrate) in the plasma are determined by the Griess calorimetric method, after reduction of the nitrate to nitrite with nitrate reductase in the following way: 12 ml of the plasma sample are mixed with 88 ml of deionized water and incubated in the dark for 1 hour at room temperature with 40 ml of phosphate buffer (0.31M, pH 7.5), 20 ml of xcex2-NADPH (reduced nicotinamide adenine dinucleotide phosphate) (0.86 mM), 20 ml of FDA (flavin adenine dinucleotide) (0.11 mM) and 20 ml of nitrate reductase (2 U/ml) (Boehringer Mannheim). 10 ml of ZnSO4 (1M) are added to precipitate the proteins, and, after mixing, the samples are centrifuged at 20,000xc3x97g for 5 minutes. Finally, 50 ml of the supernatant are incubated for 10 minutes at room temperature with 100 ml of the Griess reagent (1% sulfanilamide in a phosphoric acid/0.1% naphthylethylenediamine mixture in deionized water (V/V)). The optical densities are read at 540 nm with a microplate spectrophotometer, each point being determined twice. KNO3 and NaNO2 are used as standards for the colorimetric method.
In this test, the oligosaccharides according to the invention inhibit the formation of NOx by more than 50%.
Moreover, the oligosaccharides of formula (I) increase the survival and growth of motoneurons and are therefore particularly useful in preventing and/or treating motoneuron diseases such as amyotrophic lateral sclerosis, progressive spinal atrophy, infantile muscular atrophy and primary lateral sclerosis.
It is known that motoneuron cultures die by apoptosis if they are prepared in the absence of a trophic support (BDNF, NT5, for example). It has now been found that the oligonucleotides according to the invention enable motoneurons to survive and grow. This activity was assayed on cocultures of astrocytes and of motoneurons deprived of neurotrophic factors, according to the following protocol:
Cultures Enriched in Motoneurons:
The cultures enriched in motoneurons are prepared using the centrifugation method described by R. L. Schnaar and A. E. Schaffner, J. Neurosci., 1, 204-217 (1981) and modified by W. Camu and C. E. Henderson, J. Neurosci. Methods, 44, 59-70 (1992). Spinal cords of rat E15 embryos are sterilely dissected and the dorsal spinal notochords are removed. They are then cut up and incubated for 15 minutes at 37xc2x0 C. in PBS (phosphate buffer saline: 137 mM NaCl, 2.68 mM Kcl, 6.45 mM Na2HPO4, 1.47 mM KH2PO4) to which 0.05% of trypsin has been added. The dissociation of the cells is completed by trituration with the end of a 1 ml pipette in the culture medium supplemented with 0.1% of bovine serum albumin (BSA) and with 0.1 mg/ml of DNAase. The cell suspension layered out onto a band of 6.5% weight/volume metrizamide in L15 medium (sold by Gibco BRL) and centrifuged at 500 g for 15 minutes. The band at the interface containing the motoneurons is recovered, diluted in L15 medium and incubated for 45 minutes at room temperature in culture dishes pre-coated with anti-mouse IgG and with MC192 hybridoma supernatant (Chandler C E et al., J. Biol. Chem., 259, 6882 (1984)). The suspended cells are washed with L15 medium and the motoneurons are eluted, with gentle stirring, with the MC192 hybridoma supernatant. The motoneurons are plated out at a density of 650 cells per 24 mm in culture dishes, on astrocyte monolayers, in L15 medium to which sodium bicarbonate (22 mM), coalbumin (0.1 mg/ml), putrescine (0.1 mM), insulin (5 xcexcg/ml), sodium selenite (31 nM), glucose (20 mM), progesterone (21 nM), penicillin (100 IU/ml) and streptomycin (100 xcexcg/ml) have been added. The cultures are maintained at 37xc2x0 C. in a humidified atmosphere at 5% of CO2.
Culturing of Spinal Cord Astrocytes:
The astrocytes are obtained from rat embryos according to the method of R. P. Saneto and J. De Vellis, in Neurochemistry, a practical approach (A. J. Turner and H. S. St John) IRL Press, Oxford-Washington DC, pp. 27-63 (1987), slightly modified. The spinal cords are sterilely dissected and the meninges and dorsal ganglia are removed. Five to ten spinal cords are transferred into PBS (phosphate buffer saline: 137 mM NaCl, 2.68 mM Kcl, 6.45 mM Na2HPO4, 1.47 mM KH2PO4) and cut up before being incubated at 37xc2x0 C. for 25 minutes in PBS to which 0.25% of trypsin has been added. The enzymatic treatment is stopped by adding 10 ml of Dubelco-Eagle modified medium (DMEM) to which 10% of foetal calf serum (FCS) has been added, and the cells are collected by centrifugation. Another mechanical dissociation step is carried out using the end of a 1 ml pipette. The cells are plated out at a density of 1.5xc3x97106 cells per 25 cm2 of culture medium in DMEM containing 10% of FCS. After 2 days in vitro, the cultures are fed every day throughout the duration of the study. When a visible monolayer of cells is obtained, the cells are shaken for 48 hours at 250 rpm and, the following day, the monolayers are treated with cytosine arabinoside (10xe2x88x925 M) for 48 hours. The astrocyte monolayers are then amplified to a density of five per 35 mm on culture plates, for 25 cm2 culture flasks at the start of the study.
The spinal astrocyte cultures are composed of more than 98% cells which are immunoreactive for glial fibrilary acidic protein (GFAP).
The astrocyte monolayers are exposed either to PBS alone (controls) or to the product to be tested in solution in PBS for 24 hours at the concentration of 0.1 ng/ml to 10 ng/ml. The astrocyte monolayers are then washed with DMEM and maintained for 2 hours with culture medium to which the motoneurons are added. Two hours after feeding, and for 2 to 3 days, the vehicle or the product to be tested is again added to the culture medium.
Immunochemical Identification of the Motoneurons:
The cells are fixed in 4% of paraformaldehyde and 0.1% of glutaraldehyde in PBS (pH 7.4 at 4xc2x0 C. for 15 minutes). The cultures are then washed and the nonspecific sites are blocked with 2% of bovine serum albumin (BSA) in PBS and 0.1% of Triton X100(copyright). These cultures are successively incubated with p75LNGRF antibodies (article by Chandler, cited above), overnight at 4xc2x0 C., and with biotinylated goat serum (1/125, Gibco) and streptavidin-peroxidase (1/200, Gibco) for 60 minutes. The antibodies are visualized using the DAB/hydrogen peroxide reaction.
Cell Counting and Statistical Analysis
The cells which are immunoreactive for the low activity neutrophin receptor p75lngfr and which exhibit neurites longer than the diameters of 4 cells are considered to be viable motoneurons. The number of motoneurons is evaluated by counting the labelled cells in a surface area of 0.825 cm2 under a microscope with a 200 fold magnification. The values are expressed as the number of motoneurons per cm2 or a percentage of the number of motoneurons present in the cultures maintained in the absence of trophic factor compared to the control. The experiments are carried out at least 3 times.
The statistical analyses are carried out using the Student""s test (t-test).
Using pretreatments with the oligosaccharides of the present invention, the number of motoneurons which grow on the astrocyte monolayer is increased from 20 to 50%.
The following examples are representative of the preparation of the oligosaccharides of formula (I) and of the intermediates.
In these examples, the abbreviations have the following meanings:
xcex94Is: (4-deoxy-2-O-sulfo-xcex1-L-threo-hex-enopyranosyluronic acid)-(1xe2x86x924)-2-deoxy-2-sulfoamino-6-O-sulfo-xcex1-D-glucopyranose, tetrasodium salt, or xcex94UAp2S-(1xe2x86x924)-xcex1-D-GlcNp2S6S
Is: (2-sulfo-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2-deoxy-2-sulfoamino-6-O-sulfo-xcex1-D-glucopyranose, tetrasodium salt, (2-sulfo-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2-deoxy-6-O-sulfo-2-sulfoamino-xcex1-D-glucopyranose, tetrasodium salt, or xcex1-L-idoAp2S-(1xe2x86x924)-xcex1-D-GlcNp2S6S
IIs: (xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2-deoxy-6-O-sulfo-2-sulfoamino-xcex1-D-glucopyranose, trisodium salt, or xcex1-L-idoAp-(1xe2x86x924)-xcex1-D-GlcNp2S6S
IIIs: (2-sulfo-xcex1-L-idopyranosyluronic acid)-(1xe2x86x924)-2-deoxy-2-sulfoamino-xcex1-D-glucopyranose, trisodium salt, or xcex1-L-idoAp2S-(1xe2x86x924)-xcex1-D-GlcNp2S
IdoAp: idopyranosyluronic acid
GlcNp: 2-amino-2-deoxyglucopyranose
xcex94Uap: 4-deoxy-xcex1-L-threo-hexenopyranosyluronic acid
S: sulfate.